ArticleMethods and protocols2022
A Robust Expression and Purification Method for Production of
Article in Methods and protocols, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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Who cites it
5 citing papers in PubMed.
- A galactose-based auto-expression system improves T7-inducible protein production in Escherichia coli.Scientific reports · 2025Article
- Simplified Protocol for the Purification of Native Cas Nucleases for DNA-Free Genome Editing.Methods and protocols · 2025Article
- Optimization of a CRISPR-Cas9Frontiers in plant science · 2025Article
- Optimizing Recombinant Cas9 Expression: Insights fromBiomedicines · 2024Article
- An Efficient Expression and Purification Protocol for SpCas9 Nuclease and Evaluation of Different Delivery Methods of Ribonucleoprotein.International journal of molecular sciences · 2024Article
Corrections and comments
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Authors and funding
3 authors.
Funding
Abstract
Genome editing using the CRISPR/Cas9 system is one of the trendiest methodologies in the scientific community. Many genome editing approaches require recombinant Streptococcus pyogenes Cas9 (SpCas9) at some point during their application, for instance, for in vitro validation of single guide RNAs (SgRNAs) or for the DNA-free editing of genes of interest. Hereby, we provide a simple and detailed expression and purification protocol for SpCas9 as a protein fused to GFP and MBP. This protocol improves protein yield and simplifies the purification process by overcoming the frequently occurring obstacles such as plasmid loss, inconsistent protein expression levels, or inadequate protein binding to affinity resins. On average, this protocol yields 10 to 30 mg of purified, active, His6−MBP−SpCas9 NLS−GFP protein. The purity addressed through SDS-PAGE is > 80%.
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Registered trials
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